Transmissible spongiform encephalopathies (TSEs) are a collection of neurodegenerative diseases characterized by progressive dementia, ataxia, amyloid plaque formation and spongiform degeneration in the central nervous system (CNS) (Prusiner, S. B., 1993, Dev. Biol. Stand. 80, 31–44). The causative agent in such diseases is now understood to be abnormal prion protein. The fundamental event in TSEs such as Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) is cattle and scrapie in sheep is the conversion of the normal cellular prion protein PrPC, into a pathogenic isoform, PrPsc. Accumulation of PrPsc in the brain of prion-infected animals correlates with the rise in titer of infectious prions and is used as a diagnostic marker for prion diseases. In light of the threat of an interspecies transmission of BSE to humans, a large number of domestic animals must be tested for the presence of PrPsc in the brain or other suitable material. In the absence of covalent modifications that would allow a distinction between PrPsc and PrPC, PrPsc is routinely detected in Proteinase K (PK)-treated homogenates by Western blotting or enzyme-linked immunosorbent assay (ELISA) utilizing the fact that PrPsc but not PrPc is partially protease resistant. Notably, these currently available assays do not take advantage of the fact that PrPsc forms aggregates. It is now believed that formation of detergent-resistant PrPsc aggregates is a general biochemical property of PrPsc even for rare prion strains where PrPsc is sensitive to proteolytic digestion. This aggregation occurs when the prions are exposed to an aggregation aid for example including a complexing agent.
The fatal human neurodegenerative disorder CJD has also been transmitted iatrogenically via a number of routes suggesting the possibility that the causative agent might also be transmitted via blood products. The identification of a new form of human TSE, named “variant” CJD (vCJD), confirmation of an association with the agent of bovine spongiform encephalopathy (BSE) and evidence that the distribution of the agent of vCJD in human tissues may differ from that of classical CJD suggests the existence of a theoretical risk that blood or blood products may transmit PrPsc (see Turner et al., Blood Reviews 1998; 12:255–68).
A number of blood products are prepared for medical use from pooled donations of human plasma including normal and specific immunoglobulins, coagulation factor concentrates and solutions of albumin. There is currently considerable concern about the possibility that biopharmaceutical products from human or animal sources may transmit TSEs. Human plasma proteins for parenteral administration inherently carry a risk for disease transmission. Current technology for plasma screening and process steps for the removal or inactivation of viruses has greatly improved the safety of these products, see Burnouf T, et al., Blood Reviews 2000; 14:94–110, in this regard. However, suitable screening tests have not yet been developed for abnormal PrPsc, which are also extremely resistant to chemical and physical means of inactivation. To determine the probability of vCJD having been transmitted to patients by products derived from this plasma, it is necessary to determine the transmissibility of the PrPsc in clinically relevant circumstances, the extent to which procedures used for plasma fractionation were capable of eliminating the PrPsc from plasma products, and the extent to which the agent PrPsc can be detected in the biological product using available assays.
Human plasma is obtained from whole blood following removal of the larger cellular fractions. Recent studies performed by the plasma fractionation industry have demonstrated that process steps used in the manufacture of human plasma products may reduce PrPsc (see Foster P., Trans. Med. 1999, 9:3–14; Lee DC et al., J. Virol. Methods 2000, 84:77–89; Foster P. et al., Vox Sang 2000, 78:86–95, and Lee D. et al., Transfusion 2001, 41:449–55.) These process steps include Cohn fractionation, depth filtration and chromatography. Foster et al. (Vox Sang, supra.) demonstrated that depth filtration was effective in removing significant amounts of abnormal prion protein (PrPsc) from both immunoglobulin and albumin.
There is therefore a need to develop methods of capture and removal of the abnormal infective prions from animal or human derived medicinal products or food products which are effective yet do not substantially degrade and/or remove the biological activity or food value of the product. Due to the limitations of the current methods of detection and quantitation of abnormal prions, there is an unmet need in ability to concentrate to above detection limits and thereafter detect and accurately quantitate the abnormal prion protein (PrPsc) from the sample.
The instant invention is based on the surprising discovery that depth filtration of aqueous liquids containing biological products, such as for example a biologically active protein, with one or more depth filters having a pore size less than six microns, is surprisingly effective in removing abnormal infective prion proteins. More particularly, these inventors have made the surprising discovery that depth filtration of aqueous liquids containing biological products, such as for example a biologically active protein, with one or more depth filters having a pore size less than six microns, after treatment with an aggregation aid, is surprisingly effective in removing abnormal infective prion proteins.
The invention provides a method for the capture, removal, concentration and subsequent accurate quantitation of PrPsc associated with TSEs, when such TSEs are contained in biological or food products.
In particular, the invention provides a method for said capture, removal, concentration and subsequent accurate quantitation of PrPsc associated with TSEs, in biologicals that have been treated with one or more aggregation aids which results in aggregation of the PrPsc such that the PrPsc will be captured in and on a filter. Any method that results in such aggregation may be employed as an aggregation aid as contemplated herein. In particular it has been found that solvents such as for example alcohols may be employed. In the methods of the invention, an aggregation aid such as a solvent liquid that has been admixed with the biological or food product is passed through a filter formed of a matrix of cellulose fiber impregnated with diatomaceous earth or similar filter material which may be coated with a cationic resin having an average pore diameter of the filter ranging from 0.1 micron to 6 micron. Typically the filter may be a single use disposable filter.
In particular, the invention provides a method for the capture, removal, concentration and subsequent accurate quantitation of PrPsc associated with TSEs in biologicals that have been treated with one or more aggregation aids, for example solvent such as for example an alcohol, such as for example alcohol-fractionated immunoglobulin solutions, which comprises passing the solvent liquid containing the biological or food product through a depth filter formed of a matrix comprising solid particles of porous material and having a pore size providing a retention less than 6 μm. Typically the filter will be a single use disposable filter. The treatment with the aggregation aid(s) may be accomplished with the one or more aids admixed together or used in series.
By the terms “removal” or “capture” is meant the actual physical removal of the PrPsc from the liquid containing the desired protein. For practical purposes, the recovery of the desired protein in its original biological state should be substantially maintained at least to a level in excess of 50%, preferably 80%, more preferably >90%.
Using the methods of the invention, removal of the abnormal infective prion protein may be achieved to an extent of at least 102.5, 103, preferably 104, more particularly >105.
Aside from removal of the infective PrPsc from the biological or food product, the invention also relates to the elution from the one or more filters and subsequent concentration of the captured and eluted PrPsc using an elution buffer which may comprise, for example, hypertonic solutions such as for example high salt solutions so the PrPsc may be accurately quantitated using available assays.
Thus, the instant invention provides for aggregation of prions followed by filtration for the purification of a biological or food solution, the elution of the prions from the filter and the concentration of the PrPsc so as to enable one skilled in the art to employ available assays to quantitate both total prion and PrPsc in a biological or food sample. The invention will further allow the rapid high-throughput testing of large numbers of samples for PrPsc.
The invention also relates to the treated biological or food solution.
Since the source of human plasma is whole blood following removal of the larger cellular fractions, we therefore, in order to simulate the state expected of a TSE agent in plasma for fractionation, herein used as an inoculum a fraction of scrapie-infected hamster brain from which intact cells and larger fragments had been removed. TSE diseases are believed to be transmitted either by protease -K-resistant, conformationally abnormal prion protein (PrPsc). We herein disclose an in vitro method of analysis to determine the distribution of hamster-adapted scrapie PrPsc as a marker for the partitioning behavior of vCJD.
TSE agents are highly resistant to inactivation, therefore reduction of any product-associated risk will be dependent on the physical removal of infective material during product manufacture. Process technologies used in the manufacture of plasma products include the separation of proteins by precipitation and chromatography with resultant protein solutions being clarified and sterilized by depth and membrane filtration procedures, respectively. Some of these technologies by their modification with the methods of this invention, may be capable of removing TSE agents from a product stream.
PrP protein was detected herein using a Western Blot with the monoclonal antibody 3F4 specific for hamster PrP. This antibody reacts with residues 109–112 PrP from only humans, hamsters and felines. Incubation with 3F4 antibody was at a concentration of 0.6 ug/ml for a minimum of 1 hour, after which excess antibody was washed away and the membranes incubated with a rabbit anti-mouse horseradish peroxidase conjugate (1:1000 dilution) for a minimum of 1 hour. After extensive washing with TTBS, the membranes were developed using enhanced chemiluminescence.
In the manufacture of RhoGAM® RHO(D) Immune Globulin (Human) by this Assignee, PrPsc was removed to the limit of detection during depth filtration steps that are also used in the manufacture of immunoglobulins.
Western blotting is a method used to identify and characterize PrPsc. The PrPsc is isolated by extraction and is differentiated by its partial resistance to proteinase K digestion. The PrPRES (PrPsc resistant to proteinase digestion) is identified by the migration positions of the glycosylation forms and fragments. The sensitivity of this assay is approximately 3 logs less sensitive than the infectivity assay. This sensitivity issue is partially overcome by centrifuging the enzyme digested preparation, removing the supernatant and resuspending the prion material in a smaller volume, resulting in a concentration of the prion material. We have shown that the prions can be easily concentrated by filtering them through a filter after treatment with an aggregation aid, and later collected in a small volume by elution. This technique can be used on a large scale to remove prions from a product stream.
This procedure will have a major impact on the use of the Western blot and indeed any other prion detection assay, to determine the presence of PrPsc in a biological matrix. This invention allows the TSE material to be quantitatively concentrated quickly to allow for enhanced detection. When seeking to purify a biological, food or cosmetic solution of PrPsc, this invention has the advantage in the ease in which the biological, food or cosmetic solution filters through the large nominal pore size of the filter.
The methods of the invention are useful for the treatment of biologicals, foods and cosmetics by removing, eluting and, further, quantitating PrPsc, and depending on the aggregation aid(s) employed, PrPC. Among the biologicals that can be so treated are blood and blood components such as whole blood, blood serum and plasma, urine, cerebrospinal fluid and blood-derived biological products such as antibodies and immunoglobulins. One such antibody is the IgG immunoglobulin known as monoclonal anti-D immunoglobulin or RhoGAM® Rho(D) Immune Globulin (Human). This polyclonal immunoglobulin is used in the prevention of hemolytic disease of newborn wherein the mother is injected with Rho(D) immunoglobulin of human origin. Such a product is RhoGAM®, available from the assignee hereof, and it operates by preventing the unimmunized Rho (D) negative mother from responding to Rho (D) antigen present on red cells and ‘received’ from an Rho(D) positive infant. Thus, by preventing anti-Rho (D) production by the mother, the subsequent Rho (D) positive infant of this mother is protected from hemolytic disease of the newborn. This successful product is currently produced by a Cohn alcohol fractionation type process.
RhoGAM® Rho(D) Immune Globulin (Human) was the first successful prophylactic use of specific antibody to achieve antibody mediated immune suppression. RhoGAM® is an IgG immunoglobulin solution containing anti-Rho(D) at a dose of 300 micrograms of anti-D activity per dose. RhoGAM® can be given to the nonimmunized, Rho(D) negative pregnant woman at the appropriate time prevent future disease in her Rho(D) positive offspring. The disease is called hemolytic disease of the newborn or more specifically, Rh-erythroblastosis fetalis.
A smaller dose of anti-Rho(D), MICRhoGAM® Rho(D) Immune Globulin (Human) Micro-Dose (50 micrograms of anti-Rho(D)) is also sold by the Assignee hereof for treatment of women who have abortions and miscarriages at twelve weeks gestation or earlier. While the full dose protects the recipient for up to 15 ml of Rho(D) positive red cells, the smaller dose provides protection up to 2.5 ml of Rho(D) positive red cells. RhoGAM® is used as antenatal prophylaxis at 26 to 28 weeks gestation. Other indications include threatened abortion at any stage of gestation with continuation of pregnancy, abortion or termination of pregnancy at or beyond 13 weeks gestation, abdominal trauma or genetic amniocentesis, chorionic villus sampling (CVS) and percutaneous umbilical blood sampling (PUBS).
Most immunoglobulin injectable materials approved for use by the FDA and Bureau of Biologics have been produced by the alcohol fractionation procedure developed by Dr. E. Cohn of Harvard during the 1940s and described in Cohn et al., J. Am. Chem. Soc. 68, 459 (1946), incorporated herein by reference. This procedure coupled with the careful selection of plasma negative for hepatitis infectivity, HIV, and other blood-borne pathogens determined by the most sensitive tests available. That the products produced by this procedure are indeed safe can easily be demonstrated by the millions of non-infected recipients of product. The inventors hereof have now found that the alcohol employed in the Cohn process referenced hereinabove is sufficient to act as an aggregation aid in that it causes sufficient numbers of PrPsc particles to aggregate, such that PrPsc can be removed to the limits of detection using the inventive depth filtration, and eluted and concentrated to a level sufficient for such detection.
The solvent composition employed has minimal effect on the IgG particle but sufficiently aggregates the PrPsc sufficient to enable it to be removed to below its level of detection using available assays.
It is therefore an object of this invention to provide a method for removal of PrPsc and if desired, PrPC, from biological and food solutions using prion aggregation aids and membrane or depth filtration. Depth filtration is preferably used.
It is also an object of the invention to remove PrPsc and if desired, PrPC, from protein-containing liquids, particularly those derived from human plasma, without unacceptable effects on the nature or biological activity of the proteins.
It is a further object of the invention to capture, concentrate and detect to accurate quantitation, PrPsc from any biological fluid using the methods disclosed herein.
It is an object of the instant invention to provide abnormal infective prion -cleared, pure immunoglobulin for injection. Such a substantially pure product is produced using the processing methods of the invention.
It is a further object of this invention to provide a manufacturable process for purifying immunoglobulins from abnormal infective prion which is reasonable in terms of temporal, square foot and protein yield requirements.
It is a further object of the invention to provide a depth filter which can be a single use filter and may be disposed of having removed PrPsc from the process stream.
It is a further object of this invention to provide a concentrated PrPsc solution, by elution of said prions from the depth filter and filter washes.
It is yet a further object of this invention to provide a rapid assay for the assessment of PrPsc in various biological materials including biological fluids and human blood and plasma-derived products. Use of such assays as, for example, the Western Blot, require sufficient levels of prions unavailable in non-prion-aggregated, non-filtered biological solutions. This method provides a practical method to capture, elute and concentrate prions so that they can be detected using currently available assays. Use of these novel capture and elution methods increases sensitivity about 3 logs, enabling reduction in the volumes needed to perform the detection assays.